1. Field of the Invention
The field of this invention relates generally to the family of CR-LR proteins, more specifically to the oligodenerocyte-myelin glycoprotein (OMgp). This invention also relates to the nucleic acid coding for the OMgp, and to segments thereof. Methods of producing the OMgp and the associated nucleic acid segments are also provided. Applications and advantages of the CR-LR protein family are presented. This invention also concerns methods to detect the OMgp and associated nucleic acid segments in specimens, such detection having as one goal to identify carriers of the neurofibromatosis gene on chromosome 17. Detection may be achieved by use of a kit.
2, Description of Related Art
A. Central Nervous System Proteins and Myelination
There are large numbers of proteins expressed in cells of the nervous system. Some of these are believed to participate in one of the major dramas of central nervous system development, myelination. Myelination is believed to involve complex interactions between oligodendrocytes and axons (Bunge, et al., 1978). Although the nature of these chemical and physical interactions has not been established, hypotheses have been proposed to explain certain portions of the myelination process. For example, Quarles has proposed that myelin-associated glycoprotein may mediate interactions between axons and myelin-forming cells (1984). Gulcher et al. (1986) relates a protein called gp 150/225 as having a similar role.
Central nervous system (CNS) myelin has a structure which is a complicated set of closely opposed oligodendrocytic membranes. Proteolipid protein and the myelin basic proteins are believed to contribute toward maintaining the opposition of compact myelin membranes, (Lemke, 1988), while the myelin-associated glycoprotein is believed to play a role in mediating the initial oligodendrocyte-neuron adhesion in periaxonal regions (Poltorak, et al., 1987).
What is known about the protein composition of myelin suggests that a few major proteins that account for most of the total protein. On a quantitative basis, there are minor proteins (Lees and Brostoff, 1984) whose functions are not known. One possible role for these minor proteins is to mediate interactions between oligodendrocytes and axons.
Proteins of the central nervous system may be associated with carbohydrates. Carbohydrates may be of functional significance by conferring specific properties on their associated proteins. (Sharon and Lis, 1982) Carbohydrates may also serve to identify proteins having specific functions. In this respect, carbohydrates are markers. An example of a protein which is postulated to play an important role in cell adhesion, and its carbohydrate component includes the HNK-1/L2 carbohydrate on the myelin-associated glycoprotein reported by McGarry et al., 1983. Carbohydrates also serve as markers for the neural cell adhesion molecule (N-CAM) (Kruse, et al., 1984), the L.sub.1 glycoprotein (Kruse, et al., 1985), the J.sub.1 glycoprotein (Kruse, et al, 1985), cytotactin (Grumet, et al., 1985), the neuron-glial cell adhesion molecule (Tucker, et al., 1984; Thiery, et al., 1985), and gp 150/225 (Gulcher, et al., 1986). These cells are likely to play important roles in cell adhesion.
B. Neurofibromatosis
One of the most common genetic diseases affecting the human nervous system is neurofibromatosis (NF) an inherited disease which is characterized by tumor formation in the nervous system. There are at least two clinically distinct forms of the disease, Type 1 (von Recklinghausen neurofibromatosis, incidence 1/3,000) and 2 (bilateral acoustic neurofibromatosis, incidence 1/100,000). Our discussion will focus on Type 1. NF1 primarily affects glial cells in the peripheral nervous system but can also affect glial cells in the central nervous syustem (CNS). The gene has nearly complete penetrance but highly variable expressivity and about 50% of cases are due to new mutations. The clinical features of neurofibromatosis include cafe-aulait spots, neurofibromas that increase in size and are the main cause of morbidity in the disease, iridial hamartomas (Lisch nodules), bone abnormalities, and learning disabilities. NF1 patients have an increased risk for developing optic gliomas as well as sarcomas which most often arise in proximity to plexiform neurofibromas. Depending on the location,number, and size of the tumors, those afflicted with NF may be disfigured, mentally retarded, or medically impaired. NF may be lethal.
The pattern of inheritance for NF is that of an autosomal dominant gene. That means that only one allele (condition of a gene) of a pair needs to be the NF gene to produce the disease. Males and females are at equal risk to inherit (carry) the gene. Families in which the gene is segregating will show affected members in successive generations.
The NF gene is not expressed in all carriers to the same degree. Because of the variable expressivity of the gene in causing the NF phenotype, some cases may go undetected. A rule of thumb is to look for cafe au lait spots in persons not obviously affected with tumors. Cafe au lait spots are pigmented patches of skin that occur in persons with the NF gene. However, cafe au lait spots are not specific for this disease. Clinical criteria for the diagnosis of NF1 have recently been agreed on by an NIH Consensus Panel (Barker et al., 1987). An examination is said to be positive for NF if there are two or more of the following features: six or more cafe au lait spots; two or more neurofibromas or one plexiform neurofibroma; freckling in the axillary or ingunal regions; optic glioma; two or more Lisch nodules (iris homartomas); a distinctive osseous lesion such as spheroid dysplasia or thinning of the long-bone cortex, with or without pseudoarthrosis; and a first degree relative who meets the above criteria for NF-1. These criteria are obviously not precise, in particular in pediatric patients. Another problem is that spontaneous mutations to NF1 will be underdetected or detected late, due to absence of a family history.
More accurate methods for the detection of persons who are at risk of NF1, but who do not show obvious symptoms such as tumor formation, are needed. It is important to diagnose such carriers because knowledge of whether or not they are carrying the gene may affect their reproductive choices. Also carriers should be monitored more than non-carriers for tumor growth to facilitate earlier surgical intervention and treatment. Prenatal testing for NF1 would also be possible if detection was not dependant on clinical symptoms.
C. Chromosomal Location of the Neurofibromatosis Gene
Some studies indicate that the NF1 gene is on chrosomome 17. Genetic linkage analysis has demonstrated that NF1 is the result of a mutation of a gene located in the proximal part of the long arm of chromosome 17. Further support for this localization was gained from the study of two unrelated families in which balanced chromosomal translocations involving chromosome 17 at band q11.2 segregate with the disorder. Although definitive proof is lacking, it is believed that these translocations directly involve and alter the NF1 locus (NF1). There have been some families with neurofibromatosis in which other chromosomal translocations cosegregate. By studying the cosegregation of these two characteristics, it was determined that their inheritance was correlated. One translocation was between Chromosomes No. 1 and 17. (A translocation results from breakage of at least 2 chromosomes with rejoining of the resulting segments in combinations not present originally.)
Further confirmation of the location of the NF1 gene came from in situ hybridization studies. In this method, nucleic acid probes are labelled with radioactive isotopes or fluorescent dyes, and applied to cells in metaphase, a stage of cell division in which the chromosomes are most readily visible under the microscope as entities. The concentration of the label indicate the region of the gene.
Problems Addressed by this Invention
There is an undisputed need for direct DNA-based testing for the NF1 mutation or its product to overcome the limitations of clinical diagnostic criteria. Ideally, the NF1 gene needed to be cloned and sequenced, although indirect tests by use of DNA markers associated with the NF1 gene are of some use. (Ward, et al., 1990; Rouleau, et al., 1990) Some information on application of markers is provided by mapping studies (O'Connell, et al., 1989), however, there are many limitations and inaccuracies using markers. One of the limitations of using markers to detect gene carriers is that at least 2 family members known to be affected must be available. These techniques, therefore, are not available for population screening for carriers of NF1. Improved methods of detection would find immediate use in genetic counseling, prenatal diagnosis, presymptomatic or asymptomatic diagnosis, and investigation of the mechanisms of genetic control of the disease to explore prevention and treatment of the clinical problems. This invention relates to a protein and its corresponding nucleic-acid sequences that have dramatic implications for diagnosing and understanding NF1.